All of the enzymes in purine biosynthetic pathway, with the exception of the purB gene product, have been sequenced, overexpressed and the proteins purified to homogeneity. Using a combination of chemical and biosynthetic methods all the substrates in this pathway with the exception of the chemically unstable intermediate phosphoribosyl amine (PRA) can be obtained in modest amounts. These results set the stage for a variety of studies to be undertaken in the next funding period: (1) Genetic and kinetic methods will be utilized to investigate the importance of channeling of chemically unstable intermediates in the purine biosynthetic pathway, both in vivo and in vitro. (2) A variety of methods including rapid chemical quench kinetics and PIX will be utilized to investigate the mechanisms of a number of the enzymes involved in this pathway: PRPP-amidotransferase, GAR synthetase, FGAR-amidotransferase, AIR synthetase. (3) AIR carboxylase has been purified to homogeneity and in contrast to expectations based on genetic studies, is the purE gene product and not the gene product of both purE and purK. Attempts will be made to define the function of purK gene product.